Tobacco use remains a major health problem despite widespread knowledge of the damaging consequences. Although current smoking cessation therapies, including nicotine replacement, bupropion, and varenicline, have had some success, they are inadequate in that only a fraction (30-40%) of smokers who try these drug treatments abstain from tobacco use. Consequently, more effective or additional aids are needed. Nicotine in tobacco smoke is generally regarded as the major compound that is responsible for the addictive properties of smoking. The reinforcing effects of numerous drugs of abuse, including nicotine, are partially modulated by dopaminergic neurotransmission (Koob and LeMoal, 1997; Di Chiara, 2000; Koob, 2000; Dani and De Biasi, 2001; Nestler, 2002; Wise, 2008). Cholinergic systems have been shown to modulate dopamine (DA) release by activating nicotinic acetylcholine receptors (nAChRs) (Dani and Di Biasi, 2001; Kaneko et al., 2000; Mameli-Engvall et al, 2006; McKay et al, 2007; Exley et al, 2008; Exley and Cragg, 2008; Deng et al, 2007; Maskos, 2008). Studies from our group (Cui et al, 2003; Salminen et al, 2004, 2007, Grady et al, 2007; Drenan et al, 2008; Grady et al, 2009b) as well as others (Kulak et al, 1997; Kaiser et al, 1998; Zhou et al, 2001; Champtiaux et al, 2003; Perry et al, 2007; Sidhpura et al, 2007; Exley et al, 2008; Perez et al, 2008, 2009; Meyer et al, 2008) indicate that a6-nAChRs (a6a4B2B3, a6B2B3, a6B2) and a4-nAChRs (a4B2, a4a5B2) nAChRs play important roles in modulating nicotine-induced increases in dopamine release. The a6B2* receptors are highly sensitive to activation by smoking concentrations of nicotine when measured in synaptosomal DA release experiments (Champtiaux et al, 2003; Salminen et al., 2004 and 2007) and dominate nicotine control of dopamine neurotransmission in the nucleus accumbens (Exley et al., 2008) These results, coupled with recent findings that polymorphisms in the human genes that code for the subunits that make up nAChRs expressed in DA neurons, a4, a5, a6, B2, B3 (CHRNA4 (Li et al., 2005, 2008; Hutchison et al, 2007) CHRNAS (Amos et al, 2008; Berrettini et al, 2008; Beirut et al. 2008; Hung et al, 2008; Schlaepfer et al, 2008; Saccone et al, 2009; Wang et al, 2009; Weiss et al, 2008; Thorgeirsson et al, 2008), CHRNA6 (Hoft et al, 2009; Saccone et al, 2009) CHRNB2 (Ehringer et al, 2007) and CHRNAB3 (Hoft et al, 2009; Saccone et al, 2009) influence vulnerability to tobacco addiction and/or lung cancer, suggest that developing drugs that modulate the nAChRs that play vital roles in modulating DA release may provide new, more effective approaches to treat tobacco addiction. It is possible that a selective agonist or antagonist for the a6B2* class of receptors could be of use as a therapy for smoking cessation. Progress toward finding such a compound was a goal of our initial collaboration. In the past few years with funding from this grant, we initially screened 16 nicotinic compounds, supplied by our collaborators at Targacept. These compounds were assayed systematically in mouse brain tissues that naturally express the nAChR of interest using a hierarchical approach to determine affinity, potency, and efficacy at four classes of nAChRs, a4B2*, a7, a3B4* and a6B2* using membrane binding assays, synaptosomal function assays, and current recordings from cells expressing a7-nAChR (this last assay was carried out at Targacept). This screen has helped our NCDDG to define modifications to the structure of nicotine to guide the synthesis of more selective compounds. Publication of this work is in progress (submitted). A second round of 6 compounds was synthesized by chemists at Targacept based on what we learned from our assays of the initial set of compounds. These provided us with 6 pairs of compounds (pyridine vs pyrimidine) to investigate our hypothesis that the pyrimidine compounds may be more selective for a6B2*-nAChR. This study has been published (Breining et al, 2009), and will help guide synthesis of the third round of compounds. We plan to assay these in a similar manner, and hope to choose a few (~3-4) for studies of behavior and chronic treatment. In addition this grant has supported research into the function of the a6B2*-nAChR. A gain-of-function mutation, a6L9'S, has been generated by our collaborators in the Lester lab at Caltech. Characterization of these mutant mice revealed that they do indeed have greatly increased activity of the a6B2*-nAChR, as well as having a distinct phenotype, and will facilitate study of modulation of behaviors by this subtype (Drenan et al., 2008). We intend to continue characterizing new and potentially more selective compounds, as well as study aspects of a4B2* and a6B2*-nAChR function using the a6L9'S mice. Unlike the natural agonist, ACh, nicotine is not metabolized quickly; therefore, chronic exposure to nicotine may act by a combination of activation and desensitization of nAChRs (Rose, 2007). Potential smoking cessation drugs will also be likely to promote some Project 3 (Boulder. Marks, PI) Lester, Henry A. level of desensitization. Data generated with the NCDDG grant suggests that a6B2*-nAChRs may desensitize less than a4B2*. The a4 and a6 null mutant mice and their wild-type littermates available at IBG will be valuable tools to assess selectivity, desensitization and to study other aspects of receptor function.